Fig. 5

Downregulated RNF128 promotes melanoma cell EMT via positively regulating the Wnt/β-catenin-CD44 loop. a Western blot was used to detect the expression of MAPK and Wnt signaling-related molecules in M14-shNC/M14-shRNF128 and A2058-Vector/A2058-RNF128 cells. b The TOP/FOP luciferase reporter assay was performed in M14-shNC/M14-shRNF128 and A2058-Vector/A2058-RNF128 cells, and the results showed that low level of RNF128 activated the canonical Wnt/β-catenin pathway. c Immunofluorescence staining was used to detect the subcellular localization of β-catenin in M14-shNC/M14-shRNF128 and A2058-Vector/A2058-RNF128 cells, and the results showed that low level of RNF128 promoted the nuclear transposition of β-catenin. d Invasion and colony formation assays were performed in the indicated cells after incubation with inhibitors of the ERK (AZD) and Wnt (XAV) pathways, and the results showed that Wnt pathway derived low level of RNF128-induced cell invasion and stemness. e Western blot was used to detect the expression of RNF128, EMT markers (E-cadherin, vimentin, and Snail), CD133, p-ERK, ERK, and β-catenin in the indicated cells, and the results showed that Wnt pathway derived low level of RNF128-induced cell EMT and stemness. f Invasion and colony formation assays were performed in the indicated cells, and the results showed that low level of RNF128 induced cell invasion and stemness via β-catenin. g Western blot was used to detect the expression levels of RNF128, β-catenin, EMT markers, and CD133, and the results showed that low level of RNF128 induced cell EMT and stemness via β-catenin. h Western blot was used to detect the expression of RNF128 and β-catenin in the indicated cells, and it showed that low level of RNF128-induced Wnt pathway activation was decreased by CD44 and CTTN knockdown. i Western blot (left) and qRT-PCR (right) were used to detect the expression of target genes of Wnt/β-catenin signaling, and the results showed that low level of RNF128 promotes the expression of CD44 and c-Myc. j Western blot (left) and qRT-PCR (right) were used to detect the expression of CD44 and c-Myc in the indicated cells, and the results showed that downregulated RNF128 induced CD44 and c-Myc expression via Wnt/β-catenin signaling. h A schematic model of the positive feedback of RNF128-Wnt signaling-CD44 axis during melanoma progression.^**p < 0.01, ^***p < 0.001, ^****p < 0.0001